| Objective: "Storing blood" is defined as the function of the liver to store,regulate,and collect blood,which is not only related to blood circulation,but also related to the function of the liver governing and smooth qi flow.When the liver’s qi is not running smoothly,the blood circulation will blocked,even become blood stasis.Clinical and experimental studies of liver depressive syndrome have found abnormalities in hemorheology and coagulation in it.Based on the relationship between liver storing blood and liver depressive syndrome,this thesis explored the changes of hemorheology and coagulation in rats with liver depression.Combining the theory of "correlation between formula and syndrome" to observe Chaihushugansan’s effect-Classic formula for liver depressive syndrome of hemorheology and coagulation and explore the related mechanism on liver depressive model.Research 1:The status of blood circulation in rats with liver depressive syndrome Methods: Forty male Wistar rats were randomly divided into control group（20 rats）and model group（20rats）.The model group was restrained for 3hours every day,8:00-11:00am for 28 days;the normal group was kept normally.On the 15 th day and 29 th day,10 rats were randomly selected from each group.The rats were given anesthesia by intraperitoneal injection of 1% pentobarbital,and blood was taken through the abdominal aortaand remove the hypothalamus,liver and other tissues.Observed indicators:（1）indicators related to liver depressive syndrome: appearance characterization and behavioral indicators: weekly observe and record the changes of each group of rats in appearance（activity,emotional,sleep,diet,stool and hair）and weight.The sugar indtake test and the open field test were carried out on the 15 th day and the29 th day.Laboratory indicators of liver depressive syndrome: plasma serotonin（5-HT）,corticosterone（CORT）,hypothalamic norepinephrine（NE）.（2）Coagulation related indicators:hemorheology,prothrombin time（PT）,activated partial thromboplastin time（APTT）,thrombin time（TT）,fibrinogen（FIB）,platelet aggregation rate.Results:（1）Indicators of liver depressive syndrome: Compared with the normal group,the model group rats from the first week to the fourth week of activity,emotional,sleep,hair,diet and stool scores were significant elevated（P<0.01）,body weight decreased significantly（P<0.01 or0.05）;sucrose consumption rate decreased significantly at the end of week2 and week4（P<0.O01 or0.05）;the total distance of crawling and the number of crossings increased significantly（P<0.01）,the residence time in the central area decreased significantly(P<0.05 in the end of second week;and the retention time in central area,total crawling distance and number of crossings were all decreased significantly in the end of fourth week（P<0.01 or0.05）.Compared with the normal group,the plasma 5-HT level at the end of the second and fourth week were significantly lower（,P<0.01 or 0.05）in model group.There was no significant change in plasma CORT level at the end of week2（P>0.05）and a significant increase at the end of week4(（P<0.05）in model group.At the end of week2,there was no significant change in NE level in the hypothalamus（P>0.05）,and it decreased significantly in the week4（P<0.01）in model group.（2）Circulation-related indicators: compared with the normal group,the blood viscosity and plasma viscosity of the model group at week2 did not change significantly（P>0.05）;whole blood viscosity at medium and high shear and plasma viscosity was significantly increased（P<0.01）at the week 4.Compared with the normal group,the PT,APTT,and TT of the model group were significantly decreased（P<0.01）,FIB was significantly increased（P<0.05）at week 2;PT and APTT decreased significantly（P<0.01）,FIB increased significantly（P<0.05）,TT decreased but the difference was not significant（P>0.05）at the week 4.Compared with the normal group,f the model group platelet aggregation rate at 3mim was significantly increased（P<0.01）at week 2;the platelet aggregation rate was significantly increased at 1mim,3min,and 5min at week4.(（P<0.01）.The results showed that at the 2nd week the liver depression rats with chronic stagnation stress gradully appeared abnormal symptoms and liver depression-related indicators,similar to those of clinical TCM liver depressive syndrome.An abnormality in blood rheology and coagulation also occurs.At the fourth week,blood rheology,PT,APTT,FIB,and platelet aggregation rate were significantly aggravated,showing a hypercoagulable state.It is speculated that with the prolongation of chronic restraint time,the liver depressive syndrome gradually aggravates and affects the "storing blood" function of the liver,which is characterized by abnormal blood rheology and hypercoagulability.Research 2: Effect of Chaihu Shugan powder on abnormal blood circulation and coagulation function in rats with liver depressive syndromeMethods: Fifty male Wistar rats weighing 210-220 g were randomly divided into control group,model group,Chaihu Shugan Powder high dose group（CH）,Chaihu Shugan Powder low dose group（CL）and fluoxetine group,with 10 rats in each group..The normal group was not treated,and the other groups were restrained for3 hours every day,8:00-11:00 am,and continuously molded for 4 weeks.After 2 weeks of modeling,the Chaihu Shugan Sangao high and low-dose groups were intragastrically administered with 1.24g/kg·d and 0.62g/kg·d Chaihu Shugan solution,and fluoxetine group was administered with 0.18mg/kg·d fluoxetine solution once a day for14 days.Rats in the normal group and the model group were given the same amount of normal saline.On the 29 th day of the experiment,the rats were anesthetized by intraperitoneal injection of 1%pentobarbital,and blood was collected from the abdominal aorta and the liver and hypothalamic tissues were remove.Results:（1）Indicators of liver depressive syndrome:Compared with the normal group,the model group rats from the first week to the fourth week of activity,emotional,sleep,hair,diet and stool scores were significant elevated（P<0.01）,body weight decreased significantly（P<0.01）;sucrose consumption rate decreased significantly at the end of week 4（P<0.05）.Compared with the model group,the activity,emotional,sleep,hair,diet and stool scores of all drug-administered group were significantly lower at the end of week4（P<0.01）.Compared with the model group,the body weight of CH group increased significantly in the third week（P<0.01）,and the body weight of CL group and CH group increased significantly at the fourth week（P<0.01or0.05）.There was no significant difference in the body weight of fluoxetine group.At the 4th week,the sucrose consumption rate in the CH group and the fluoxetine group was significantly increased（P<0.05）,and there was no significant difference of sucrose consumption rate in the CL group.Compared with the normal group,in the model group plasma5-HT and hypothalamic NE levels were significantly decreased（P<0.01）,hypothalamic CRH and plasma CORT levels were significantly increased（P<0.01）,and hypothalamic DA level was no significant difference,and observed that liver began to appear pathological damage.Compared with the model group,in the CL group plasma 5-HT and hypothalamic DA levels were significantly increased（P<0.05）and liver pathological damage was alleviated;in CH group the levels of plasma 5-HT and hypothalamic NE were significantly increased（P<0.05）,and the hypothalamus CRH and plasma CORT levels were significantly decreased（P<0.O01or0.05）,and liver pathological damage was alleviated;in the fluoxetine group Plasma5-HT level was significantly increased（P<0.01）,plasma CORT level was significantly lower（P<0.05）,and liver pathological damage was alleviated.（2）Blood-related indicators: Compared with the normal group,the blood viscosity and plasma viscosity of the model group at week2 did not change significantly（P>0.05）;whole blood viscosity at medium and high shear and plasma viscosity was significantly increased（p< 0.01）at the week4.Compared with the normal group,the whole blood viscosity at low,mediurm and high shear,plasma viscosity,erythrocyte sedimentation rate（ESR）and Equation K value of ESR of the model group were significantly increased（P<0.01）,and the blood reduced viscosity was significantly increased at different shear rates（P<0·01 or0.05）.Compared with the model group,in CL group the plasma viscosity,hematocrit,ESR,Equation K value of ESR,whole blood viscosity and whole blood reduction viscosity were significantly lower at different shear rates（P<0.01）.Compared to the model group,in CH group theESR,Equation K value of ESR,the plasma viscosity at low and medium shear rates,and the reduced viscosity of whole blood at the high and low shear rates were significantly reduced（P<0.01）.Compared with the model group,the whole blood viscosity and reduced viscosity,plasma viscosity,ESR and Equation K value of ESR of the fluoxetine group were significantly decreased（P<0.01）.Compared with the normal group,the PT and APTT of the model group were significantly decreased（P<0.01）,the FIB of the model group was significantly increased（P<0.05）,the TT of the model group difference was not significant（P>>0.05）,and the platelet aggregation rate of the model group was significantly increased at each time point（P<0.01）.Compared with the model group,in CL group the PT and APTT were significantly increased（P<0.01）,the FIB was significantly decreased（P<0.01）,and the platelet aggregation rate was significantly decreased（P<0.01）;in CH group PT and APTT were significantly increased（P<0.01）,the FIB decreased significantly（P<0.01）,the platelet aggregation rate decreased significantly（P<0.01）;the fluoxetine group PT,APTT,TT increased significantly（P<0.01 or0.05）,FIB was significantly decreased（P<0.01）,and the platelet aggregation rate was significantly decreased（P<0.01）.Coagulation mechanism related indicators:Compared with the normal group,plasma levels of6-keto-PGF1α,PC,FPS,AT-Ⅲ and t-PA in the model group were significantly decreased（P<0.01）,and plasma PAI-I and D-D were elevated,but the trend was not significant;platelet5-HT2 A,SERT,tGase,Ca2+,plasma Ps,TXB2 were significantly increased（P<0.01）.Compared with the model group,in the CL group plasma levels of 6-keto-PGF1α,PC,FPS,AT-Ⅲ,t-PA,and D-D were significantly increased（P<0.01）,plasma TXB2 was significantly decreased（P<0.01）,and platelet SERT,tGase was significantly reduced（P<0.05）;in CH group plasma 6-keto-PGFla and FPS were significantly increased（P<0.01）,platelet5-HT2 A,SERT,tGase,Ca2+,plasma Ps and TXB2 were significantly decreased（P<0.01 or 0.05）;in fluoxetine group,the plasma FPS was significantly increased（P<0.05）,and the platelet 5-HT2 A,SERT,tGase,Ca2+,plasma Ps and TXB2 were significantly decreased（P<0.01 or 0.05）.The results showed that the high and low doses of Chaihu Shugan and fluoxetine had different effects on the symptoms and indexes of TCM liver depressive syndrome in model rats with chronic restraint stress.Among them,the improvement effect of Chaihu Shugan high doses is the most obvious one.All the drug-administered groups had an improvement effect on the coagulation state of model rats.Among them,the low dose of Chaihu Shugan had a better effect on the anti-coagulant cofactor.The high dose of Chaihu Shugan and the fluoxetine group had no significant effect on fibrinolysis cofactor,but the effect on 5-HT pathway was better Conclusions: The results suggest that the effects of different doses of Chaihu Shugan are different.The high dose of Chaihu Shugan has a wider range of effects than fluoxetine in relieving the symptoms of liver depression and blood abnormal.The results also showed that the mechanism of Chaihu Shugan Powder may involve inhibiting platelet 5-HT pathway-induced platelet activation and promoting liver endothelial fibrinolysis and anticoagulant cofactor activation in model rats.This study enriched the connotation of liver depressive syndrome,especially from the perspective of blood circulation and coagulation,providing an experimental evidence for the relationship between liver depressive syndrome and liver "storing blood".It provides a certain pharmacological basis for Chaihu Shugan Powder on the regulation of hemorheology and coagulation that accompany with liverdepressive syndrome,and its clinical use in related diseases.The molecular mechanism of Chaihu Shugan Powder regulating coagulation abnormality under the condition of liver depressive syndrome also provides an experimental proof for understanding the correlation between formula and syndrome. |
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